The present invention relates to recombinant DNA molecules, and in particular to recombinant vectors for use in the transformation of a microbial host which contain inserted DNA fragments carrying one or more genes coding for one or more enzymes involved in the biosynthesis of P-Lactam antibiotics, especially penicillins and cephalosporins.
Progress in understanding the biosynthesis of xcex2-lactam antibiotics produced by micro-organisms such as Streptomyces clavuligerus has been slow. Nevertheless it has been established that the biosynthetic pathways of certain penicillins and cephalosporins (including cephamycins) are closely related.
Isopenicillin N is an intermediate in the biosynthesis of both groups of compounds and is formed by the action of a xe2x80x98cyclasexe2x80x99 enzyme on the tripeptide xcex4(L-xcex1-aminoadipyl)-L-cysteinyl-D-valine (sometimes referred to as LLD-ACV or, more simply, ACV as used hereinbelow). The intermediate isopenicillin N may be converted either into penicillin G or, by the action of an xe2x80x98epimerasexe2x80x99 enzyme, into penicillin N and it is from the latter that various cephalosporins and cephamycins may be derived by a multi-step pathway following an initial ring-expansion with an xe2x80x98expandasexe2x80x99 enzyme. A recent summary of the state of the art is given by J. F. Martin and P. Liras in Trends in Biotechnology, 1985, 3, 39-44. Thus, in the biosynthesis of Cephamycin C, penicillin N is converted into deacetoxycephalosporin C which is then converted by a dioxygenase enzyme into desacetylcephalosporin C.
The latter is O-carbamoylated to give O-carbamoyldesacetylcephalosporin C, which is then converted into Cephamycin C. It is probable, in the light of worK by J. D. Hood et al. (J. Chem.Soc., Chem. Commun, 1983, pages 1187-1188 and references therein) that the 7xcex1-methoxy group in Cephamycin C is introduced in two steps, i.e. via the action of a 7-hydroxylase enzyme on O-carbamoyldesacetylcephalosporin C to give the 7xcex1-hydroxy derivative, followed by subsequent methylation.
As is now well known, by means of recombinant DNA techniques, it is possible to insert into a host cell DNA carried on a vector with the result that the transformed host may become endowed with the capacity to synthesise whatever protein(s) or enzyme(s) the gene(s) carried on the insert DNA may encode. (For a full discussion of recombinant DNA methodology, and a glossary of the terms used therein, see xe2x80x98Principles of Gene Manipulationxe2x80x99 by R. W. Old and S. B. Primrose, 3rd Edition, Blackwell Scientific Publications, 1985).
The isolation and expression in E. coli of the isopenicillin N synthetase (cyclase) gene from C. acremonium has recently been reported by S. M. Samson et al (Nature, 1985, 318, 191-194).
In order to clearly define the invention reference is made to the accompanying drawings in which:
FIG. 1(a) is an endonuclease restriction map of S. clavuligerus ATCC 27064 chromosomal DNA (I) coding for genes involved in penicillin and cephalosporin biosynthesis;
FIG. 1(b) is an endonuclease restriction map of the portion of the DNA (I) contained in a plasmid designated pBROC 138;
FIG. 1(c) is an endonuclease restriction map of the portion of the DNA (I) contained in a plasmid designated pBROC 137;
FIG. 1(d) is an endonuclease restriction map of a portion of the DNA (I) contained in a plasmid designated pBROC 303;
FIG. 2 is an endonuclease restriction map of plasmids pBROC 137 and pBROC 138; and
FIG. 3 is an endonuclease restriction map of plasmid pBROC 303.